Detection by Western Blot Analysis of BBD126 in Protein Preparations from Bovine Sperm

Sperm cells from three different bulls were harvested from the epididymal fluid by centrifugation with a Ficoll gradient. Cells were lysed using lysis buffer: 50 mM Hepes, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 10% glycerol, 0.5% NP40, aprotinin (5 μg/ml), leupeptin (5 μg/ml), phenylmethane sulfonyl fluoride (1 mM), and Na₃VO₄ (1 mM). Protein levels of whole cell lysate were measured using a bicinchoninic acid protein assay (Thermo Scientific), and equal amounts of protein were resolved in a 4%–12% SDS-PAGE under reducing conditions (50 mM dithiothreitol [DTT] and 3% SDS), followed by transferring to polyvinylidene difluoride (PVDF) membranes (Merck Millipore) and blocking with PBS containing 0.1% Tween-20 and 5% BSA (PBST). Membranes were blotted with 1:1000 dilution of the a-BBD126 to a final concentration of 1 μg/ml solution in PBST overnight at 4°C. An anti-mouse IgG labeled with IRDye 680RD (1 mg/ml) secondary antibody was used to detect bound antibody in a 1:10 000 dilution in PBST. The membrane was analyzed using the infrared Odyssey imager (LI-COR). To validate the antibody specificity, 30 μg of total protein from cell lysate and media samples of HEK293 cells transfected with pcDNA3.1-Thio-126 BBD126 or pcDNA3.1-LacZ vectors using GeneJuice Transfection Reagent (Millipore) following the manufacturer's recommended protocol were loaded onto 4%–12% SDS-PAGE gels. After transfer into a PVDF membrane, it was blotted using 1:1000 dilution of the a-BBD126 to a final concentration of 1 μg/ml solution in PBST overnight at 4°C. The membrane was then processed as previously described.